Small molecules targeted to the microtubule-Hec1 interaction inhibit cancer cell growth through microtubule stabilization.

Highly expressed in cancer protein 1 (Hec1) is a subunit of the kinetochore (KT)-associated Ndc80 complex, which ensures proper segregation of sister chromatids at mitosis by mediating the interaction between KTs and microtubules (MTs). HEC1 mRNA and protein are highly expressed in many malignancies as part of a signature of chromosome instability. These properties render Hec1 a promising molecular target for developing therapeutic drugs that exert their anticancer activities by producing massive chromosome aneuploidy. A virtual screening study aimed at identifying small molecules able to bind at the Hec1-MT interaction domain identified one positive hit compound and two analogs of the hit with high cytotoxic, pro-apoptotic and anti-mitotic activities. The most cytotoxic analog (SM15) was shown to produce chromosome segregation defects in cancer cells by inhibiting the correction of erroneous KT-MT interactions. Live cell imaging of treated cells demonstrated that mitotic arrest and segregation abnormalities lead to cell death through mitotic catastrophe and that cell death occurred also from interphase. Importantly, SM15 was shown to be more effective in inducing apoptotic cell death in cancer cells as compared to normal ones and effectively reduced tumor growth in a mouse xenograft model. Mechanistically, cold-induced MT depolymerization experiments demonstrated a hyper-stabilization of both mitotic and interphase MTs. Molecular dynamics simulations corroborate this finding by showing that SM15 can bind the MT surface independently from Hec1 and acts as a stabilizer of both MTs and KT-MT interactions. Overall, our studies represent a clear proof of principle that MT-Hec1-interacting compounds may represent novel powerful anticancer agents.

p53 localization at centrosomes during mitosis and postmitotic checkpoint are ATM-dependent and require serine 15 phosphorylation.

We recently demonstrated that the p53 oncosuppressor associates to centrosomes in mitosis and this association is disrupted by treatments with microtubule-depolymerizing agents. Here, we show that ATM, an upstream activator of p53 after DNA damage, is essential for p53 centrosomal localization and is required for the activation of the postmitotic checkpoint after spindle disruption. In mitosis, p53 failed to associate with centrosomes in two ATM-deficient, ataxiatelangiectasia-derived cell lines. Wild-type ATM gene transfer reestablished the centrosomal localization of p53 in these cells. Furthermore, wild-type p53 protein, but not the p53-S15A mutant, not phosphorylatable by ATM, localized at centrosomes when expressed in p53-null K562 cells. Finally, Ser15 phosphorylation of endogenous p53 was detected at centrosomes upon treatment with phosphatase inhibitors, suggesting that a p53 dephosphorylation step at centrosome contributes to sustain the cell cycle program in cells with normal mitotic spindles. When dissociated from centrosomes by treatments with spindle inhibitors, p53 remained phosphorylated at Ser15. AT cells, which are unable to phosphorylate p53, did not undergo postmitotic proliferation arrest after nocodazole block and release. These data demonstrate that ATM is required for p53 localization at centrosome and support the existence of a surveillance mechanism for inhibiting DNA reduplication downstream of the spindle assembly checkpoint

p53 displacement from centrosomes and p53-mediated G1 arrest following transient inhibition of the mitotic spindle.

Growing evidence indicates a central role for p53 in mediating cell cycle arrest in response to mitotic spindle defects so as to prevent rereplication in cells in which the mitotic division has failed. Here we report that a transient inhibition of spindle assembly induced by nocodazole, a tubulin-depolymerizing drug, triggers a stable activation of p53, which can transduce a cell cycle inhibitory signal even when the spindle-damaging agent is removed and the spindle is allowed to reassemble. Cells transiently exposed to nocodazole continue to express high levels of p53 and p21 in the cell cycle that follows the transient exposure to nocodazole and become arrested in G(1), regardless of whether they carry a diploid or polyploid genome after mitotic exit. We also show that p53 normally associates with centrosomes in mitotic cells, whereas nocodazole disrupts this association. Together these results suggest that the induction of spindle damage, albeit transient, interferes with the subcellular localization of p53 at specific mitotic locations, which in turn dictates cell cycle arrest in the offspring of such defective mitoses.

Binding properties, cell delivery, and gene transfer of adenoviral penton base displaying bacteriophage.

The penton base of adenovirus mediates viral attachment to integrin receptors and particle internalisation, properties that can be exploited to reengineer prokaryotic viruses for the infection of mammalian cells. We report that filamentous phage displaying either the full-length penton base gene or a central region of 107 amino acids on their surface were able to bind, internalise, and transduce mammalian cells expressing integrin receptors. Both phage bound alphavbeta3, alphavbeta5, alpha3beta1, and alpha5beta1 integrin subtypes. Cell-binding was shown by electron microscopy; internalisation was investigated by immunofluorescence and confirmed by micropanning. As it has been described for adenovirus, pharmacologic disruption of phosphoinositide-30H kinase, but not of myosin light-chain kinase, inhibited phage internalisation. Recombinant phage encoding an eukaryotic expression cassette was able to mediate gene expression in mammalian cells. Taken together, these data open insights for the exploit of recombinant phage for integrin-targeted gene delivery.

Normal and cancer-prone human cells respond differently to extremely low frequency magnetic fields.

Human lymphoblastoid cells of normal origin and from genetic instability syndromes, i.e. Fanconi anemia (FA) group C and ataxia telangectasia, were continuously exposed to extremely low frequency magnetic field (ELF-MF). We report that ELF-MF, though not perturbing cell cycle progression, increases the rate of cell death in normal cell lines. In contrast, cell death is not affected in cells from genetic instability syndromes; this reflects a specific failure of the apoptotic response. Reintroduction of complementation group C in FA cells re-established the apoptotic response to ELF-MF. Thus, genes implicated in genetic instability syndromes are relevant in modulating the response of cells to ELF-MF.

Regulated Ran-binding protein 1 activity is required for organization and function of the mitotic spindle in mammalian cells in vivo.

Ran-binding protein (RanBP) 1 is a major regulator of the Ran GTPase and is encoded by a regulatory target gene of E2F factors. The Ran GTPase network controls several cellular processes, including nucleocytoplasmic transport and cell cycle progression, and has recently also been shown to regulate microtubule nucleation and spindle assembly in Xenopus oocyte extracts. Here we report that RanBP1 protein levels are cell cycle regulated in mammalian cells, increase from S phase to M phase, peak in metaphase, and abruptly decline in late telophase. Overexpression of RanBP1 throughout the cell cycle yields abnormal mitoses characterized by severe defects in spindle polarization. In addition, microinjection of anti-RanBP1 antibody in mitotic cells induces mitotic delay and abnormal nuclear division, reflecting an abnormal stabilization of the mitotic spindle. Thus, regulated RanBP1 activity is required for proper execution of mitosis in somatic cells.

Engagement of CD4 before TCR triggering regulates both Bax- and Fas (CD95)-mediated apoptosis.

In the present study, we have aimed at clarifying the CD4-dependent molecular mechanisms that regulate human memory T cell susceptibility to both Fas (CD95)-dependent and Bcl-2-dependent apoptotic pathways following antigenic challenge. To address this issue, we used an experimental system of viral and alloantigen-specific T cell lines and clones and two ligands of CD4 molecules, Leu-3a mAb and HIV gp120. We demonstrate that CD4 engagement before TCR triggering suppresses the TCR-mediated neosynthesis of the Flice-like inhibitory protein and transforms memory T cells from a CD95-resistant to a CD95-susceptible phenotype. Moreover, evidence that the apoptotic programs were executed while Fas ligand mRNA expression was inhibited led us to analyze Bcl-2-dependent pathways. The data show that the engagement of CD4 separately from TCR influences the expression of the proapoptotic protein Bax independently of the anti-apoptotic protein Bcl-2, whereas Ag activation coordinately modulates both Bax and Bcl-2. The increased expression of Bax and the consequent dissipation of the mitochondrial transmembrane potential (DeltaPsim) suggest a novel immunoregulatory function of CD4 and demonstrate that both passive cell death and activation-induced cell death are operative in CD4+ memory T cells. Furthermore, analysis of the mechanisms by which IL-2 and IL-4 cytokines exert their protective function on CD4+ T cells in the presence of soluble CD4 ligands shows that they were able to revert susceptibility to Bax-mediated but not to CD95-dependent apoptotic pathways.

Induction of polyploidy and apoptosis after exposure to high concentrations of the spindle poison nocodazole.

The proportions of aneuploid/polyploid versus euploid cells formed after treatment with spindle poisons like nocodazole are of course dependent on the relative survival of cells with numerical chromosome aberrations. This work aimed at studying the survival of polyploid cells formed after treatment with a nocodazole concentration sufficient to significantly decrease tubulin polymerization (0.1 microg/ml). First, normal primary lymphocytes were analysed and the following complementary chromosomal parameters were quantified: mitotic index, frequency of abnormal mitoses, polyploid metaphases and apoptotic cells. The results clearly indicate a positive correlation between abnormal mitotic figures, apoptosis and the induction of polyploidy. They therefore led to a single cell approach in which both apoptosis and polyploidy induction could be scored in the same cell. For this purpose, actively proliferating cells are required and two human leukaemic cell lines were used, KS (p53-positive) and K562 (p53-negative), which have a near-triploid karyotype. Cells were separated into an apoptotic and a viable fraction by means of annexin-V staining and flow cytometry. In KS, treatment with nocodazole induced a similar fraction of hexaploid cells in both the viable and apoptotic fraction, but no dodecaploid cells were ever observed. In contrast, a population of dodecaploid cells (essentially viable) was clearly observed in the K562 cell line. The results in KS, as compared with K562, confirm that wild-type p53 can prevent further cycling of polyploid cells by blocking rereplication. The most probable explanation for these data is that not only the mitotic spindle but also interphase microtubules are sensitive to nocodazole treatment. Our data thus strongly suggest that besides the G(1)/S checkpoint under the control of p53, the G(2)/M transition may be sensitive to depolymerization of microtubules, possibly under the control of Cdc2, Bcl-2, Raf-1 and/or Rho.

p53-independent apoptosis and p53-dependent block of DNA rereplication following mitotic spindle inhibition in human cells.

We have studied the response of human transformed cells to mitotic spindle inhibition. Two paired cell lines, K562 and its parvovirus-resistant KS derivative clone, respectively nonexpressing and expressing p53, were continuously exposed to nocodazole. Apoptotic cells were observed in both lines, indicating that mitotic spindle impairment induced p53-independent apoptosis. After a transient mitotic delay, both cell lines exited mitosis, as revealed by flow-cytometric determination of MPM2 antigen and cyclin B1 expression, coupled to cytogenetic analysis of sister centromere separation. Both cell lines exited mitosis without chromatid segregation. K562 p53-deficient cells further resumed DNA synthesis, giving rise to cells with a DNA content above 4C, and reentered a polyploid cycle. In contrast, KS cells underwent a subsequent G1 arrest in the tetraploid state. Thus, G1 arrest in tetraploid cells requires p53 function in the rereplication checkpoint which prevents the G1/S transition following aberrant mitosis; in contrast, p53 expression is dispensable for triggering the apoptotic response in the absence of mitotic spindle.

TCR engagement regulates differential responsiveness of human memory T cells to Fas (CD95)-mediated apoptosis.

In this work, we have tried to establish whether human memory T cells may be protected from Fas (CD95)-induced apoptosis when correctly activated by Ag, and not protected when nonspecifically or incorrectly activated. In particular, we wanted to investigate the molecular mechanisms that regulate the fate of memory T cells following an antigenic challenge. To address this issue, we chose an experimental system that closely mimics physiological T cell activation such as human T cell lines and clones specific for viral peptides or alloantigens. We demonstrate that memory T cells acquire an activation-induced cell death (AICD)-resistant phenotype when TCRs are properly engaged by specific Ag bound to MHC molecules. Ag concentration and costimulation are critical parameters in regulating the protective effect. The analysis of the mechanisms involved in the block of CD95 signal transduction pathways revealed that the crucial events are the inhibition of CD95-associated IL-1beta-converting enzyme (ICE)-like protease (FLICE) activation and poly(ADP)-ribose polymerase cleavage, and the mRNA expression of FLICE-like inhibitory protein. Furthermore, we have observed that TCR-mediated neosynthesis of FLICE-like inhibitory protein mRNA is suppressed either by protein tyrosine kinase inhibitors or cyclosporin A. In conclusion, the present analysis of the effects of TCR triggering on the regulation of AICD suggests that AICD could be inhibited in human memory T cells activated in vivo by a foreign Ag, but may become operative when the Ag has been cleared.

Dexamethasone induces apoptosis in human T cell clones expressing low levels of Bcl-2.

Previous results of ours have demonstrated that the same clonotype can express both a sensitive and a resistant phenotype to Dex-mediated PCD induction depending on its cell cycle phase. In particular, we demonstrated that human T lymphocytes, arrested in the G0/G1 phase of the cell cycle, are susceptible, while proliferating T cells are resistant to Dex-mediated apoptosis. In this paper, we have further characterized the sensitive and resistant phenotypes and investigated whether a different expression of the apoptotic genes Fas, FasL, Bcl-2, Bcl-x and Bax is involved in the regulation of Dex-mediated apoptosis. The results show that the amount of Bcl-2 expression, that changes during cell cycle phases, determines susceptibility or resistance to apoptosis induced by Dex. In fact, undetectable expression of Bcl-2 in sensitive cells favors Dex-mediated apoptosis while high expression of Bcl-2 in proliferating cells counterbalances apoptosis induction. Moreover, the addition of exogenous IL-2, in the presence of Dex, fails to up-regulate Bcl-2 expression and to revert Dex-mediated apoptotic phenomena.

Phyllanthus orbicularis aqueous extract: cytotoxic, genotoxic, and antimutagenic effects in the CHO cell line.

The present work evaluates the cytotoxic, genotoxic, and antimutagenic effects of Phyllanthus orbicularis (plant of genus Phyllantus) aqueous extract in Chinese hamster ovary (CHO) cells. P. orbicularis aqueous extracts are used in Cuban traditional medicine for their antiviral activity against Hepatitis B virus and A and B flu virus. The cytotoxicity of the extract was tested by means of colony-forming ability and growth-inhibition assays as well as by measuring the mitotic index. Apoptosis induction and cell-cycle kinetics were analyzed by cytofluorimetric methods. Chromosome aberration assays were performed to study the genotoxic and antimutagenic activity of the extract. Results show that doses of up to 100 microg/ml of the extract did not induce any cytotoxic effects. Cell survival and mitotic index decreased significantly at doses higher than 100 microg/ml as a function of dose as well as of treatment time. Moreover, continuous treatments of up to 18 h induced the appearance of a significant number of apoptotic cells. Following a 3-h exposure to a dose of 750 microg/ml, cells accumulated significantly in G(2)-M phase and remained blocked in G(1-) and G(2)-M phases after several posttreatments in fresh growth medium. The aqueous extract alone did not induce chromosome aberrations but, in combined treatment with H(2)O(2), significantly reduced H(2)O(2)-induced chromosome aberrations. Flow cytometric analysis of DCFH intracellular oxidation showed that the extract decreased the oxidizing power of H(2)O(2.) This ability could possibly explain the extract's antigenotoxic activity. Absence of cytotoxicity at the lower tested doses and the antimutagenic properties of the extract stimulate the interest in studying possible new pharmaceutical uses of P. orbicularis.

Towards a unifying model for the metaphase/anaphase transition.

The term mitosis actually covers a complex sequence of events at the level of the cell membrane, the cytoplasm, the nuclear membrane and the chromosomes; recently attention has been focused more and more on the checkpoints that control their orderly progression. The term 'checkpoint' refers here to the inhibitory pathways that coordinate coupling between the sequence of events, ensuring dependence of the initiation of each upon successful completion of others. This paper will mainly focus upon the possible checkpoint which controls a brief but essential step, dissociation of the sister chromatids into two identical chromosomes. This step will be called the metaphase/anaphase transition. First, the molecular components that are important in metaphase/anaphase transition will be reviewed: accurate segregation of sister chromatids between the daughter cells is dependent on coordinated interaction of centrosomes, centromeres, kinetochores, spindle fibres, topoisomerases, proteolytic processes and motor proteins. Deficiencies in or impairment of any of these structures or in their control systems may lead to a more or less important genomic imbalance. A model combining the ultrastructural components, the molecular components and the controlling molecules will be proposed. The unifying concept emerging from this synthesis indicates that sister chromatids separate independently of the tubulin fibres, as a result of proteolytic processes controlled by the anaphase promoting complex. The spindle fibres are thus necessary to move the separated chromatids to the spindle poles but probably not to initiate separation. A number of remaining questions are also highlighted.

DNA damage and cytotoxicity induced by beta-lapachone: relation to poly(ADP-ribose) polymerase inhibition.

beta-Lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5, 6-dione) was previously shown to enhance the lethality of X-rays and radiomimetic agents and its radiosensitizing role in mammalian cells was attributed to a possible interference with topoisomerase I activity. Furthermore, beta-lapachone alone was found to induce chromosomal damage in Chinese hamster ovary (CHO) cells. The aim of the present study was to further elucidate the possible mechanisms by which beta-lapachone exerts its genotoxic action in cultured mammalian cells. Flow cytometry analysis of beta-lapachone-treated CHO cells indicated a selective cytotoxic effect upon S phase of the cell cycle. beta-lapachone produced DNA strand breaks as determined by alkaline elution assay; alkaline elution profiles from treated cells showed a bimodal dose-response pattern, with a threshold dose above which a massive dose-independent DNA degradation was observed. Furthermore, beta-lapachone increased the capacity of crude CHO cellular extracts to unwind supercoiled plasmid DNA, while significantly inhibiting in vitro poly(ADP-ribose) polymerase (PARP). These results suggest that damage induction is probably mediated by the interaction between beta-lapachone and cellular enzymatic function(s), rather than reflecting a direct action on the DNA. We suggest that the inhibition of PARP plays a central role in the complex biological effects induced by beta-lapachone in CHO cells.

Deregulated expression of the RanBP1 gene alters cell cycle progression in murine fibroblasts.

RanBP1 is a molecular partner of the Ran GTPase, which is implicated in the control of several processes, including DNA replication, mitotic entry and exit, cell cycle progression, nuclear structure, protein import and RNA export. While most genes encoding Ran-interacting partners are constitutively active, transcription of the RanBP1 mRNA is repressed in non proliferating cells, is activated at the G1/S transition in cycling cells and peaks during S phase. We report here that forced expression of the RanBP1 gene disrupts the orderly execution of the cell division cycle at several stages, causing inhibition of DNA replication, defective mitotic exit and failure of chromatin decondensation during the telophase-to-interphase transition in cells that achieve nuclear duplication and chromosome segregation. These results suggest that deregulated RanBP1 activity interferes with the Ran GTPase cycle and prevents the functioning of the Ran signalling system during the cell cycle.

The in vitro micronucleus test: a multi-endpoint assay to detect simultaneously mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction.

Genotoxicity testing aims to detect a large range of genetic damage endpoints and evaluate such results in context of cell survival. The cytokinesis block micronucleus test offers the advantage to provide simultaneously information on both cell cycle progression and chromosome/genome mutations. Indeed, 1. frequencies of cytokinesis-blocked binucleated cells (and polynucleated) are good estimators of the mitotic rate; 2. frequencies of apoptotic figures in mononucleated and binucleated cells provide a measure for cell death before or after cell division; 3. combination of fluorescence in situ hybridization (FISH) for centromere/telomeres and micronucleus scoring allows discrimination between clastogenic and aneugenic events; 4. detection of FISH signals for chromosome specific sequences in both macronuclei and micronuclei, discriminates between aneuploidy due to chromosome non-disjunction or to chromosome loss. The cytokinesis block in vitro micronucleus test is thus a cytogenetic multi-test providing mechanistic information with a simple, rapid, objective, microscopical analysis.

Deregulated apoptosis is a hallmark of the Fanconi anemia syndrome.

Fanconi anemia (FA) is a genetic human disorder associated with bone marrow failure and predisposition to cancer. FA cells show poor growth capacity and spontaneous chromosomal anomalies as well as cellular and chromosomal hypersensitivity to DNA cross-linking agents such as mitomycin C (MMC). Because it is likely that disruption of the apoptotic control would lead to such a phenotype, we investigated the implication of apoptosis in the FA syndrome. It is shown that, although demonstrating a high frequency of spontaneous apoptosis, FA cells from four genetic complementation groups are deficient in gamma-ray-induced apoptosis and their MMC hypersensitivity is not due to apoptosis. Fas is a cell surface receptor belonging to the tumor necrosis factor receptor family and is involved in apoptosis. We show that, independently of DNA damage, the alteration in the control of apoptosis in FA concerns also the pathway initiated by Fas activation. Finally, ectopic expression of the wild-type FAC gene corrects the MMC hypersensitivity and anomalies in apoptosis and cell cycle response in FA cells. Altogether, these findings strongly implicate the FA genes as playing a major role in the control of apoptosis. Thus, further studies with FA syndrome will be instrumental toward molecularly dissecting the apoptotic pathways.

Cutting edge: CD4+ T cells kill CD8+ T cells via Fas/Fas ligand-mediated apoptosis.

To explore the possibility that CD4+ T cells, described to mediate the elimination of themselves or B lymphocytes, could also mediate the elimination of CD8+ T cells, we analyzed apoptotic phenomena in cocultures of CD4+ and CD8+ autologous T cell lines. The data show that CD8+ T cells were lysed by activated CD4+ helper T cells by a Fas/FasL-mediated mechanism. CD4+ T cells were not lysed by activated CD8+ T cells, although Fas and FasL were equally expressed and anti-Fas Abs induced apoptosis in both CD4+ and CD8+ T cell populations. The results allowed us to speculate that CD4+ T cells not only help CD8+ T lymphocytes to mature into effector killer cells and to sustain this function but can also limit their growth.

Differential susceptibility to monomeric HIV gp120-mediated apoptosis in antigen-activated CD4+ T cell populations.

To support the hypothesis that indirect mechanisms mediated by viral products like the HIV envelope glycoprotein gp120 could be responsible for T lymphocyte depletion in HIV infection, we developed a system in which the impairment of T cell functions could be investigated in vitro. In particular, we characterized the conditions that allow T lymphocytes repeatedly stimulated with an antigen to be sensitive or resistant to gp120-mediated apoptotic signals. To achieve this goal, a panel of antigen-specific CD4+ T cell clones and primary CD4+ T lymphocytes were treated for 2 and 18 h with saturating amounts of monomeric gp120 (without cross-linking with specific antibodies) and antigen-driven T cell proliferation and apoptosis were analyzed. We show that monomeric gp120 induces apoptosis only in T lymphocytes repeatedly stimulated with the antigen, that primary T lymphocytes are resistant to programmed cell death mediated by monomeric gp120, but are sensitive to anti-CD4 antibodies, and that gp120-mediated apoptosis is dependent on the period of time between the binding of gp120 to CD4 and the encounter with antigen. To investigate the different susceptibility to gp120 induced apoptosis of primary CD4+ and T cell clones further, the number of membrane CD4 molecules and their affinity for gp120, together with Bcl-2 and Fas expression, were studied. Our data suggest that a down-modulation of membrane CD4 together with high expression of the Bcl-2 gene and protein characterizes the susceptibility to apoptosis of gp120-treated cells. In conclusion, our results define the phenotypic features of T cells susceptible to HIV gp120-induced apoptosis and demonstrate that the same clonotype, depending on the activation state, may present a differential sensitivity to apoptosis induction.

Low environmental radiation background impairs biological defence of the yeast Saccharomyces cerevisiae to chemical radiomimetic agents.

Background radiation is likely to constitute one of the factors involved in biological evolution since radiations are able to affect biological processes. Therefore, it is possible to hypothesize that organisms are adapted to environmental background radiation and that this adaptation could increase their ability to respond to the harmful effects of ionizing radiations. In fact, adaptive responses to alkylating agents and to low doses of ionizing radiation have been found in many organisms. In order to test for effects of adaptation, cell susceptibility to treatments with high doses of radiomimetic chemical agents has been studied by growing them in a reduced environmental radiation background. The experiment has been performed by culturing yeast cells (Saccharomyces cerevisiae D7) in parallel in a standard background environment and in the underground Gran Sasso National Laboratory, with reduced environmental background radiation. After a conditioning period, yeast cells were exposed to recombinogenic doses of methyl methanesulfonate. The yeast cells grown in the Gran Sasso Laboratory showed a higher frequency of radiomimetic induced recombination as compared to those grown in the standard environment. This suggests that environmental radiation may act as a conditioning agent.